Biosynthesis of sialylated lipooligosaccharides in Haemophilis ducreyi is dependent on exogenous sialic acid and not mannosamine. Incorporation studies using N-acylmannosamine analogues, N-glycolylneuraminic acid, and C-13-labeled N-acetylneuraminic acid

Haemophilus ducreyi is a Gram-negative bacterium that causes chancroid, a s exually transmitted disease. Cell surface lipooligosaccharides (LOS) of H. ducreyi are thought to play important biological roles in host infection. T he vast majority of H. ducreyi strains contain high levels of sialic acid ( N-acetylneuraminic acid, NeuAc) in their LOS. Here we investigate the biosy nthetic origin of H. ducreyi sialosides by metabolic incorporation studies using a panel of N-acylmannosamine and sialic acid analogues. Incorporation of sialosides into LOS was assessed by matrix-assisted laser desorption an d electrospray ionization mass spectrometry. A Fourier transform ion cyclot ron resonance mass spectrometer provided accurate mass measurements, and a quadrupole time-of-flight instrument was used to obtain characteristic frag ment ions and partial carbohydrate sequences. Exogenously supplied N-acetyl mannosamine analogues were not converted to LOS-associated sialosides at a detectable level. In contrast, exogenous C-13-labeled N-acetylneuraminic ac id ([C-13]NeuAc) and N-glycolylneuraminic acid (NeuGc) were efficiently inc orporated into LOS in a dose-dependent fashion. Moreover, approximately 1.3 muM total exogenous sialic acid was sufficient to obtain about 50% of the maximum production of sialic acid-containing glycoforms observed under in v itro growth conditions. Together, these data suggest that the expressed lev els of sialylated LOS glycoforms observed in H. ducreyi are in large part c ontrolled by the exogenous concentrations of sialic acid and at levels one might expect in vivo. Moreover, these studies show that to properly exploit the sialic acid biosynthetic pathway for metabolic oligosaccharide enginee ring in H. ducreyi and possibly other prokaryotes that share similar pathwa ys, precursors based on sialic acid and not mannosamine must be used.